Microbial culture having catechol 1,2-oxygenase activity

ABSTRACT

This invention provides novel strains of microorganisms (e.g., Pseudomonas putida Biotype A) which are capable of converting substrates such as toluene or catechol to muconic acid quantitatively by the ortho (catechol 1,2-oxygenase) pathway. 
     Muconate lactonizing enzyme is not induced in the microorganism, thereby permitting the muconic acid to be produced and accumulated in a quantity greater than one gram of muconic acid per liter of bioconversion medium.

This patent application is a continuation of pending patent applicationSer. No. 516,088, filed July 22, 1983; now U.S. Pat. No. 4,731,328 whichis a continuation-in-part of patent application Ser. No. 287,344, filedJuly 27, 1981, now abandoned.

The present invention is related to the subject matter of U.S. Pat. No.4,355,107 which issued Oct. 19, 1982.

BACKGROUND OF THE INVENTION

Adipic acid is an important commodity in the chemical industry,particularly or consumption as a commoner in the synthesis of polymers.Adipic acid can be obtained by oxidation of cyclohexane or cyclohexanol.Another prospective method is by the hydrogenation of muconic acid,which is a diolefinically unsaturated adipic acid derivative: ##STR1##

A potentially convenient source of muconic acid is by themicrobiological oxidation of various hydrocarbon substrates.Microbiological oxidation of hydrocarbons is reviewed in AppliedMicrobiology, 9(5), 383(1961) and in "Advances in Enzymology", 27,469-546(1965) by Interscience Publishers.

U.S. Pat. No. 3,383,289 describes a process for producing amethyl-substituted muconic acid and/or 2,3-dihydroxybenzoic acid whichinvolves subjecting a C₇ -C₁₀ methylbenzene having 1-4 methyl groups andat least two consecutive unsubstituted ring carbon atoms in the presenceof a nutrient medium and under fermentation conditions to the action ofan orthodihydroxylating and nondecarboxylating strain of Nocardia.

The Journal of Biological Chemistry, 241(16), 3776 (1966) reports theconversion of catechol and protocatechuate to β-ketoadipate byPseudomonas putida. The conversion of catechol proceeds by the orthopathway via a muconic acid intermediate: ##STR2## The chemicalstructures illustrated in the reaction scheme are catechol, muconicacid, muconolactone, β-ketoadipate enollactone and β-ketoadipate,respectively.

In the Journal Of Bacteriology, 134, 756(1978) there is reported a studyof the ubiquity of plasmids in coding for toluene and xylene metabolismin soil bacteria. One of the mutant strains of Pseudomonas putidaisolated had the ability to metabolize toluene via benzyl alcohol,benzaldehyde, benzoic acid and catechol by the ortho pathway throughβ-ketoadipate to biomass and carbon dioxide.

The enzymes functioning in the toluene metabolism by the ortho pathwayincluded toluene mono-oxygenase, benzyl alcohol dehydrogenase,benzaldehyde dehydrogenase, benzoate oxygenase, dihydrodihydroxybenzoatedehydrogenase, catechol 1,2-oxygenase and muconate lactonizing enzyme.The subsequently formed β-ketoadipate was further assimilated to biomassand carbon dioxide. The mutant strains that metabolized toluene via theortho pathway did not accumulate muconic acid, since the said muconicacid metabolite was further transformed in the presence of muconatelactonizing enzyme.

No known naturally occurring microorganisms (e.g., Pseudomonas putida)are known that metabolize an aromatic substrate such as toluene by theortho pathway via muconic acid and β-ketoadipate. Wild strainsmetabolize aromatic hydrocarbon substrates by the meta pathway via2-hydroxymuconic semialdehyde instead of a muconic acid intermediate.Catechol 2,3-oxygenase is functional rather than catechol 1,2-oxygenase.

Thus, the potential of microbiological oxidation of an aromaticsubstrate such as toluene as a convenient source of muconic acidrequires the construction of mutant strains of microorganisms which (1)metabolize an aromatic substrate via catechol by means of the orthopathway, (2) allow the accumulation of muconic acid without its furtherassimilation, and (3) contain catechol 1,2-oxygenase which is notinhibited by accumulated muconic acid in a bioconversion medium.

Accordingly, it is an object of this invention to provide a process forconstruction of novel strains of microorganisms which metabolizecatechol or a catechol-precursor by the ortho pathway to accumulatedmuconic acid.

It is another object of this invention to provide novel strains ofpseudomonads which metabolize toluene or other benzoic acid-precursor tomuconic acid quantitatively, with an accumulation of greater than onegram of muconic acid per liter of bioconversion medium.

Other objects and advantages of the present invention shall becomeapparent from the accompanying description and examples.

DESCRIPTION OF THE INVENTION

One or more objects of the present invention are accomplished by theprovision of a process for the construction of novel microorganismstrains which comprises (1) culturing a microorganism speciesselectively to provide strain A1 which metabolizes an aromatic substrateselected from toluene and other catechol-precursors by the ortho pathwayvia catechol to muconic acid, and which subsequently metabolizes theresultant muconic acid via β-ketoadipate to biomass and carbon dioxide;(2) continuously and selectively culturing strain A1 for rapid growth onthe aromatic substrate as the sole source of carbon to provide strainA2; (3) culturing strain A2 in selective enrichment cycles in a mediumcontaining benzoate as the sole source of carbon and containing anantibiotic which kills only growing cells; (4) harvesting the strain A2cells and diluting and culturing the cells in media containing anon-selective carbon source; (5) plating the strain A2 cells on anutrient medium containing a limiting amount of a non-selective carbonsource and excess benzoate; (6) isolating cells from single smallcolonies, and culturing the cell isolates and selecting a strain A3,wherein strain A3 converts the aromatic substrate to accumulated muconicacid.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows muconic acid production by Pseudomonas putida BiotypeA, ATCC 31916.

The above construction procedure is generally applicable tomicroorganisms that metabolize catechol-precursor type of organicsubstrates such as ethylbenzene, styrene, benzyl alcohol orbenzaldehyde. The procedure can be modified to accommodatemicroorganisms that metabolize other aromatic substrates such asbenzene, naphthalene, phenol, salicylic acid, aniline, anthranilic acid,and the like, as disclosed ,in copending patent application Ser. No.513,231, filed July 22, 1983, now U.S. Pat. No. 4,588,688), incorporatedherein by reference.

The starting microorganism can be any organism capable of growth on theselected aromatic substrate and possessing active catechol1,2-oxygenase, e.g., a pseudomonad. A variety of naturally occurringorganisms have these traits including some members of the speciesPseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens;some members of the genera Azotobacter and Nocardia; and a number ofunclassified fungi (both molds and yeasts).

In another embodiment, this invention provides biologically puremicrobial cultures which possess catechol 1,2-oxygenase enzyme withactivity that is not inhibited in the presence of a low level of muconicacid in a bioconversion medium. A present invention microorganismconverts catechol quantitatively to accumulated muconic acid."Biologically pure" refers to a microbial culture which does not containmore than about one cell per 10⁵ cells which is not essentiallyidentical to the predominate strain cell population.

Preferred microorganisms are those which have been modified by aconstruction procedure to possess a novel combination of enzymes whichinclude catechol 1,2-oxygenase with activity that is not inhibited inthe presence of a low level up to about one gram or more of muconic acidper liter of bioconversion medium.

This invention provides microbial cultures each of which has thefollowing characteristics:

(a) possesses active benzoate dioxygenase;

(b) possesses active 1,2-dihydrodihydroxybenzoate dehydrogenase;

(c) lacks active catechol 2,3-oxygenase;

(d) does not grow on benzoate or halobenzoate;

wherein the microbial culture is capable of metabolizing catecholquantitatively to an accumulated quantity of muconic acid greater thanabout one gram per liter of a bioconversion medium.

In another embodiment, this invention provides microbial cultures eachof which exhibits the following enzymatic activities:

(a) possesses active benzoate dioxygenase and1,2-dihydrodihydroxybenzoate dehydrogenase and catechol 1,2-oxygenase;

(b) lacks active catechol 2,3-oxygenase; and

(c) does not grow on benzoate or monohalobenzoate;

wherein the microbial culture is capable of metabolizing an aromaticsubstrate selected from toluene and other catechol-precursors by theortho pathway quantitatively to an accumulated quantity of muconic acidgreater than about one gram per liter of a bioconversion medium."Quantitative" refers to a conversion selectivity to muconic acid of atleast about 90 percent.

Further illustrative of the invention microorganisms are constructedbiologically pure strains of fluorescent Pseudomonads each of which hasthe following characteristics:

(a) possesses active benzoate dioxygenase;

(b) possesses active 1,2-dihydrodihydroxybenzoate dehydrogenase;

(c) possesses catechol 1,2-oxygenase with activity that is not inhibitedin the presence of any low level quantity of muconic acid up to aboutone gram per liter of a bioconversion medium;

(d) lacks active muconate lactonizing enzyme;

(e) lacks active catechol 2,3-oxygenase;

(f) does not grow on benzoate or monohalobenzoate (e.g.,monochlorobenzoate); and

(g) cells are rod shaped, vigorously motile and polarly flagellated.

A novel strain of Pseudomonas putida Biotype A, constructed inaccordance with the present invention and having the above recitedmodified characteristics, has been deposited with the American TypeCulture Collection and has been designated as ATCC No. 31916.

In a further embodiment, this invention provides a process for theproduction of muconic acid which comprises feeding toluene or othercatechol-precursor aromatic substrate to a bioconversion mediumcontaining a novel strain of fluorescent Pseudomonas, e.g., a strainhaving an enzyme profile similar to that of ATCC No. 31916 and itsmutants.

The rate of aromatic substrate conversion typically is at least about 30milligrams of muconic acid produced per dry weight gram of cells perhour. The conversion of aromatic substrate proceeds readily at a dryweight cell concentration of 50 grams per liter, with a resultantmuconic acid production rate of 1.5 grams per liter per hour.

Under optimal conditions, the muconic acid accumulation limit canapproach up to about 50 grams of muconic acid per liter of bioconversionmedium. The microbiological oxidation process normally is conducted atambient temperatures up to about 31° C.

The ortho pathway (also known as the β-ketoadipate pathway or thecatechol 1,2-oxygenase pathway) has been studied in Pseudomomonasputida, Acinebacter calcoaceticus, and Alcaligenese eutrophus. Researcheffort for the most part has concentrated on the metabolism of benzoate.The organisms are ubiquitous in nature and are easily isolated byenrichment culture on benzoate containing media. The initial reaction inthe metabolism of benzoate is transport of the molecule into the cellfollowed by conversion of benzoate via benzoate dioxygenase and1,2-dihydrodihydroxybenzoate dehydrogenase to catechol. The series ofenzymes which convert catechol to β-ketoadipate constitute the orthopathway proper. Catechol 1,2-oxygenase is the enzyme responsible for theconversion of catechol to muconic acid as described above.

Nature 188 560(1960) describes the meta pathway (also known as the2,3-oxygenase pathway) for the metabolism of catechol and its precursorsto carbon dioxide and cell carbon. The first intermediate after catecholis the intensely yellow 2-hydroxymuconic semialdehyde. Because the ratelimiting reaction in this pathway occurs at some point after thesemialdehyde formation, the compound is excreted into the medium wheninduced cells are exposed to catechol. This phenomenon serves as a basisfor differentiating between cells using either the ortho or the metapathway.

The microorganisms growing at the expense of benzoate via the orthopathway grow at an appreciably higher rate than microorganisms growingon benzoate via the meta pathway, i.e., 50 minutes versus 210 minutesper doubling. A microorganism capable of metabolizing toluene via theortho pathway therefore appears to have a decided selective advantage.

A novel mutant strain of the present invention (e.g., Pseudomonas putidaBiotype A, strain ATCC No. 31,916) has characteristics which are uniquefor the microbiological conversion of toluene or other benzoicacid-precursor or catechol for the production and accumulation ofmuconic acid at an exceptional rate and concentration.

First, the parent microorganism is capable of growing at a rapid rate,e.g., a growth doubling time of about 1.5 hours on toluene.

Second, the mutant microorganism metabolizes toluene or other benzoicacid-precursor by the ortho pathway via catechol cleavage by the actionof catechol 1,2-oxygenase. Concomitantly, no active catechol2,3-oxygenase is induced in the microorganism culture.

Third, the catechol 1,2-oxygenase activity is not repressed or inhibitedby the presence of a low level of muconic acid, e.g., a level of up toabout one gram or more of muconic acid per liter of bioconversionmedium. This permits the accumulation of muconic acid at a level whichis higher than about one gram/liter of bioconversion medium.

Fourth, the ortho pathway series of conversion reactions is blockedsubsequent to the formation of the muconic acid from catechol. Themutant microorganism lacks the presence of active muconate lactonizingenzyme. Hence, the muconic acid is able to accumulate as it is produced,i.e., the muconic acid accumulates up to a level of about 50 grams perliter of bioconversion medium. No microorganism reported in theliterature is known to exhibit the ability to produce and accumulatemuconic acid to these levels from an aromatic substrate.

Microbial cultures provided by the present invention have an inherentgenome characteristic in common, i.e., each microbial culture is capableof bio-oxidizing catechol or a catechol-precursor by the ortho pathwayto an accumulated quantity of muconic acid in a bioconversion system.Included in the invention microbial cultures are mutant strains whichare acidophilic, i.e., they are capable of expressing their enzymaticproperties in a bioconversion medium having a pH less than about 5, suchas strains of Thiobacillus acidophillus which have been modified inaccordance with a present invention construction procedure.

Microorganism Construction Procedure

In accordance with the present invention, a procedure has been developedto isolate a strain of organism which rapidly converts toluene or otherbenzoic acid-precursor to accumulated muconic acid. The first step is toisolate a mutant preferably of an original pseudomonad type isolatewhich grows on toluene or other catechol-precursor via the orthopathway, i.e., the pathway in which muconic acid is an intermediate.

The original isolate is first made constitutive for growth on m-toluicacid. This first strain is then subjected to a cycle designed toeliminate the meta pathway and select for cells which have retained theability to grow on toluene or other selected benzoic acid-precursorsubstrate. Cells are first grown from low dilution on benzoic acid.These cells are transferred to medium containing m-toluic acid as thesole source of carbon. After one hour, the antibiotics penicillin andD-cycloserine respectively are added at concentrations of 12 and0.1/mg/ml and the incubation is continued for four to six hours. Afterthe incubation, the cells are washed and transferred at a 50:1 dilutionto medium containing the selected substrate as the sole sources ofcarbon. Visible growth occurs in approximately thirty-six hours.

When plated on agar containing benzoate a mixture of small and largecolonies are formed. Virtually all of the large colonies metabolize theselected substrate via the ortho pathway, thus producing muconic acid asan intermediate. This second strain, characterized by growth on theselected substrate via the ortho pathway, does not possess an activecatechol 2,3-oxygenase. Its doubling time on the selected substrate isapproximately two to three hours.

The second strain is then subjected to selection for a rapid growth rateby being continuously cultured on the substrate as the sole source ofcarbon. Once the culture has stabilized at a doubling time ofapproximately four hours, the dilution rate is increased to require adoubling time of three hours. This process is repeated until the cellsare growing with a doubling time of one to two hours. This third straindiffers from its parent at least in its being constitutive for catechol1,2-oxygenase.

The third strain converts the selected substrate to muconic acid butalso converts muconic acid to biomass and carbon dioxide. To obtain astrain which accumulates muconic acid, it is necessary to isolate cellslacking a functional muconate lactonizing enzyme. The third strain isgrown overnight on the selected substrate. These cells are transferredto media containing benzoic acid as the sole source of carbon. After onehour, penicillin and D-cycloserine are added and the incubation iscontinued for four to six hours. After the incubation, the cells areharvested, washed and transferred at a 500:1 dilution to mediumcontaining p-hydroxybenzoate as the sole source of carbon. Cells grownovernight on p-hydroxybenzoate are transferred to medium containingbenzoate as the sole source of carbon and the enrichment cycle isrepeated. After six cycles, the survivors are plated on agar containing5 mM benzoic acid and 0.5 mM succinic acid. On this medium, cells unableto metabolize benzoate form small colonies.

The single small colonies are picked and cultured, and after inductionwith the selected substrate, checked for their ability to producemuconate. A strain is selected which exhibits an ability to convert theselected substrate to muconic acid in an efficient manner.

The following examples are further illustrative of the presentinvention. The components and specific ingredients are presented asbeing typical, and various modifications can be derived in view of theforegoing disclosure within the scope of the invention.

The basal salts medium employed for all of the series had the followingcomposition:

50 mM of Na₂ HPO₄

100 mM of KH₂ PO₄

17 mM of (NH₄)₂ SO₄

1 mM of MgSO₄

0.1 mM of CaCl₂

0.01 mM of FeSO₄

The medium had a pH of 6.2, and the original organism used in theExamples was constructed from a natural isolate.

For cultivation, carbon sources such as toluene were added asepticallyprior to inoculation. Incubation conditions were in 250 ml shake flasks.Shaking was in a rotary shaker with temperature controlled at 28° C.

Toluene was delivered to the shake flasks either from an ethanolsterilized dialysis bag or from a 5 ml layer of paraffin wax in thebottom of the flask. In the latter case, molten paraffin was pipettedinto the flask, the flask was autoclave sterilized, and while still hot,toluene was added and mixed with the paraffin. After solidifying, thesterilized basal salts medium was added aseptically. In the case of thedialysis bags, the dialysis tubing was washed extensively and boiled toremove the glycerol which is incorporated as a plasticizer. Enoughglycerol remained to support the growth of the microorganisms to theextent of approximately 6×10⁸ cells per ml. In this system, only growthin excess of 7.5×10⁸ ml was considered significant. The basal saltsmedium was capable of supporting growth of 3.3×10⁹ cells per ml whenthere was an unlimited carbon source.

Growth was typically measured by determining the turbidity of the cellsuspension in a Klett-Summerson Colorimeter using the #66 red filter.One Klett unit was found to be equivalent to 3×10⁶ cells per ml or 17.5mg wet weight per liter of 3.5 mg dry weight per liter.

Cultures were stored under liquid nitrogen.

EXAMPLE I

This Example illustrates the isolation of toluene oxidizingmicroorganisms.

Soil samples were collected from a variety of areas and added to mediumplus paraffin containing toluene. After shaking at 28° C. for 24 hoursgrowth was apparent in the medium. Strains were isolated by streaking onagar plates containing a vial of toluene in the lid. Colonies appearedon the agar after approximately 36 hours. The size of these coloniesranged from 1 to 5 mm. A representative sampling of these colonies wastaken and cultures were stored under liquid nitrogen for long-termpreservation.

A strain derived from one of the largest colonies was chosen for furtherwork and designated MW 1000. This strain was identified as a Pseudomonasputida Biotype A on the basis of the following criteria:

(a) the cells were rod shaped, vigorously motile and polarlyflagellated;

(b) cells grew well on benzoate and p-hydroxybenzoate;

(c) cell growth on benzoate induced the synthesis of carboxymuconatelactonizing enzyme and carboxymuconolactone decarboxylase but notprotocatechuate oxygenase, a pattern of regulation characteristic onlyof the Pseudomonas putida Biotype A;

(d) the induced enzymes muconolactone isomerase, carboxy-muconatelactonizing enzyme, and carboxy-muconolactone decarboxylase wereimmunologically identical with those enzymes synthesized by Pseudomonasputida Biotype A, a saprophytic organism extensively studied in theliterature.

A growth study of MW 1000 on toluene was conducted and it was found thatthe organism grew with a doubling time of approximately 3.5 hours andhad a 5 hour lag period. Toluene grown MW 1000 consumed oxygen whenpresented with toluene, benzyl alcohol, benzaldehyde, m-toluate orcatechol, but not with benzoate. With catechol the medium turned yellowindicating the production of excess 2-hydroxymuconic semialdehyde.

The presence of the meta pathway was confirmed by demonstration of2,3-oxygenase activity in cell free extracts and a failure todemonstrate the 1,2-oxygenase. MW 1000 also oxidized benzoate via themeta pathway following induction with benzoate.

EXAMPLE II

This Example illustrates methods of constructing a strain of organismwhich oxidizes toluene via the ortho (catechol 1,2-oxygenase) pathway.

A series of mutants which metabolized toluene through the ortho pathwaywas constructed by first blocking the meta pathway and then isolatingphenotypic revertants which had reacquired the ability to grow onbenzoate. Strains possessing a meta pathway block were isolated afterpenicillin plus D-cycloserine enrichment for organisms which failed togrow on benzoate. Some fifty isolates were then spotted onto agar platesand incubated in the presence of toluene. Virtually all isolatesreverted to growth on toluene. The plates were sprayed with 10 mMcatechol and approximately 25% of the revertants were found not toproduce 2-hydroxymuconic semialdehyde. None of the colorless revertantswas found to possess an active catechol 2,3-oxygenase followinginduction with toluene.

It has been shown by Worsey and Williams, J. Bacteriol. 130, 1149 (1977)that growth on benzoate tends to cure a population of its TOL plasmidbecause the ortho pathway supports a higher growth rate. Since toluatecan only be metabolized via the meta pathway, an alternative way to curea population of its TOL plasmid is to use the penicillin plusD-cycloserine procedure to enrich for cells unable to grow on toluate.

Both these techniques were used in succession followed bycounter-selection for growth on toluene. MW 1200 was first cultured ontoluene. A small portion (0.05 ml) of this culture was transferred to 50ml of benzoate medium. After growth on benzoate the cells weretransferred to toluate and incubated for approximately one hour.Penicillin and D-cycloserine respectively were then added as describedabove and the incubation was continued for four to six hours. Cells wereharvested, washed and transferred to a toluene containing medium. Growthon toluene required approximately 36 hours indicating an exceptionallylow number of cells surviving the selection procedure.

After growth on toluene the cells were plated on benzoate agar andincubated for 48 hours, and a number of large colonies and a few smallcolonies were formed. After spraying with catechol it was found that allof the small colonies turned yellow (indicating the presence of the metapathway) but none of the large colonies did. Large colonies were pickedand cultured and it was found that, following growth on toluene, thesestrains contained no functional 2,3-oxygenase but were fully induced forthe 1,2-oxygenase. These strains metabolized toluene by the orthopathway. One isolate, designated MW 1210, was selected for further work.A growth study with MW 1210 showed a doubling time of approximately 2hours.

The procedure developed for isolation of these mutants proved to behighly repeatable. The difference in colony size between meta oxidizersand ortho oxidizers was repeatable if plates were observed at 48 hours.

The frequency of the ortho oxidizers following this procedure has rangedfrom 50 to 100% of the total colonics on the plate. Enrichment from asingle cycle, was on the order of 10⁷, although there was no means ofassessing the concentration of mutants at the individual steps.

EXAMPLE III

This Example illustrates the construction of the novel Pseudomonasputida Biotype A strain ATCC No. 31916 of the present invention.

The strain of Example II was subjected to continuous cultivation withtoluene as the sole source of carbon. Initially a dilution rate of 0.15hours⁻¹ was employed. After the culture had stabilized, the dilutionrate was increased successively to 0.25 hour⁻¹, 0.34 hour⁻¹, and 0.46hour⁻¹. An isolate was made from the cells which dominated the cultureat this latter dilution rate. This strain (MW 1211) was then used toconstruct a strain which accumulates muconic acid to greater than onegram per liter.

The above strain was cultured overnight in liquid medium on toluene asthe sole source of carbon, then benzoate was added to a level of 5 mMand the incubation was continued for approximately 1 hour. Penicillin Gand D-cycloserine were added at concentrations of 12 and 0.1 mg/mlrespectively. The antibiotic incubation was continued for approximately5 hours. The cells were then harvested by centrifugation and washedtwice with sterile de-ionized water. An aliquot of these cells was thentransferred to fresh medium containing 0.5 mM p-hydrobenzoate as a solesource of carbon, and the medium was incubated overnight. The procedurewas repeated starting with induction with benzoate.

After 6 cycles those cells present in the culture after overnight growthon p-hydroxybenzoate were diluted and plated on an agar mediumcontaining 0.5 mM succinate and 5.0 mM benzoate as sole sources ofcarbon. After 36 hours incubation the plate showed a mixture of largeand small colonies. Cells from a number of small colonies were culturedin liquid medium, induced with toluene and tested for their ability toaccumulate muconic acid. Of some 20 isolates one strain (MW 1211.1) wasan accumulator of muconic acid.

EXAMPLE IV

This Example illustrates the conversion of toluene to muconic acid withan accumulation of greater than one gram of muconic acid per liter ofconversion medium.

The microorganism employed was the ATCC No. 31916 strain of Pseudomonasputida Biotype A described in Example III.

Succinate was used as the source of carbon in the medium containing theATCC No. 31,916 culture. After reaching a stationary phase, toluene wasadded to the medium to induce the appropriate enzymes. After about 2.5hours, the cells were harvested by centrifugation and washed withbuffer.

The conversion was performed in 150 mM of sodium potassium phosphatebuffer at a pH of 7.5. The cell concentration was adjusted to 50 gm dryweight per liter. Toluene was added slowly in the vapor phase bybubbling the air or oxygen stream through a toluene reservoir. Theconcentration of muconic acid thereby produced was determinedspectrophotometrically by the increase in absorbance at 260 nm. Themuconic acid concentration rose to about 35-40 mM before the reactionbecomes inhibited as shown in the FIGURE.

The identity of the muconic acid product was confirmed by high pressureliquid chromatography, melting point, and nuclear magnetic resonance.

EXAMPLE V

This Example illustrates the bioconversion of catechol to muconic acid,employing ATCC No. 31916 strain of Pseudomonas putida Biotype A.

Cells of ATCC No. 31,916 strain were inoculated into a Chemap 10 literfermentor containing NO medium and 20 mM gluconate as a carbon source.The fermentor temperature was controlled at 29° C., and the agitationrate was 800 rpm and the air flow rate was 2.8 l/min.

After overnight incubation, the culture turbidity was 226 Klett units(0.79 g/l). At this point, feeds of acetic acid at a rate of 0.198g/l/hr and catechol (from a 1M solution) at a rate of 2.22 mM/hr werebegun. The acetic acid was added to ensure adequate carbon and energyfor maintenance and enzyme synthesis (the absence of supplemental aceticacid yielded substantially the same results). A pH of 6.4 was controlledby automatic addition of 2N NaOH.

Muconic acid synthesis initiated immediately and continued at the samerate as catechol addition. The total quantity of catechol added was 400mM and the reservoir was depleted in approximately 1023 minutes. Theconversion of catechol to muconic acid was quantitative.

EXAMPLE VI

This Example illustrates the construction of an acidophilicmicroorganism capable of converting an aromatic substrate to muconicacid under low pH conditions.

Strain A2 (MW 1211) is patch mated with an auxotrophic strain ofPseudomonas aruginosa carrying a plasmid with chromosome-mobilizingactivity, and which codes for antibiotic resistance (e.g., kanamycinresistance). Ex-conjugates are selected for growth on toluene plus 400μg/ml kanamycin. Colonies of ex-conjugates are picked and patch matedwith strain B1 (Thiobacillus acidophillus).

Ex-conjugates of this second mating are selected for growth on tolueneat a pH of 3.5. Strain B2, a colony which grows under these acidicconditions, has the ability to grow on toluene via muconic acid at lowpH. Strain B2 possesses a catechol 1,2-oxygenase which is not inhibitedby a low level up to one gram of muconic acid per liter of bioconversionmedium, and it possesses active muconate lactonizing enzyme.

In order to construct a mutant of strain B2 which is capable ofproducing and accumulating muconic acid from toluene, the same selectionprocedure is applied to Strain B2 as employed in Example III forderiving strain MW 1211.1 from strain MW 1211.

The resultant mutant obtained from the selection procedure, Strain B3,is capable of converting toluene (or other benzoic acid-precursor) at apH of less than about 5 to an accumulated quantity of muconic acidgreater than about one gram per liter of a bioconversion medium.

What is claimed is:
 1. A microbial culture comprising a microorganismhaving the following characteristics:(a) possesses active benzoatedioxygenase and 1,2-dihydrodihydroxybenzoate dehydrogenase and catechol1,2-oxygenase; (b) lacks active catechol 2,3-oxygenase; and (c) does notgrow on benzoate or monohalobenzoate;wherein the microbial culture iscapable of metabolizing an aromatic substrate selected from toluene andother benzoic acid-precursors by the ortho pathway via catecholquantitatively to an accumulated quantity of muconic acid greater thanabout one gram per liter of a bioconversion medium.
 2. Catechol1,2-oxygenase in a microbial growth culture medium wherein the catechol,1,2-oxygenase has activity that is not inhibited in the presence of lessthan about one gram per liter of muconic acid in the microbial culturegrowth medium, and wherein the microbial culture growth medium iscapable of metabolizing an aromatic substrate selected from toluene andother benzoic acid-precursors by the ortho pathway via catecholquantitatively to an accumulated quantity of muconic acid greater thanabout one gram per liter of microbial culture growth medium.